Brandi et al. BMC Medical Genetics(2020) 21:225
https://doi.org/10.1186/s12881-020-01165-0

RESEARCH ARTICLE

Open AccessWilson disease, ABCC2 c.3972C > T
polymorphism and primary liver cancers:
suggestions from a familial cluster
Giovanni Brandi1,2* , Alessandro Rizzo1, Marzia Deserti1,3, Valeria Relli2,3, Valentina Indio4, Sofia Bin2, Milena Pariali3,
Andrea Palloni1,2, Stefania De Lorenzo1,2, Francesco Tovoli5 and Simona Tavolari1,3

Abstract
Background: Polymorphisms in genes modulating xenobiotics metabolism, in particular the ABCC2 c.3972C > T
single nucleotide polymorphism (SNP) at exon 28, have been suggested to increase primary liver cancer (PLC) risk.
Conversely, the occurrence of PLCs in Wilson disease patients is a rare event, in contrast with the occurrence
observed in other chronic liver diseases. Here we report the clinical case of five siblings carrying the ABCC2
c.3972C > T SNP; three of them were affected by Wilson disease and two brothers with Wilson disease also
developed PLCs.
Methods: The presence of the ABCC2 c.3972C > T SNP was assessed by Sanger sequencing and the exposure of
PLC risk factors by standardized questionnaires.
Results: Notably, PLCs occurred only in the two brothers with the ABCC2 c.3972C > T SNP and Wilson disease who
resulted exposed to asbestos and cigarette smoking, but not in the other siblings with the ABCC2 c.3972C > T SNP,
alone or in association with Wilson disease, not exposed to these carcinogens and/or to other known risk factors
for PLCs.
Conclusions: These findings suggest that ABCC2 c.3972C > T SNP and WD, also in association, may not represent a
sufficient condition for PLC development, but that co-occurrence of further host/exogenous risk factors are neededto drive this process, reinforcing the notion that liver carcinogenesis is the result of a complex interplay between
environmental and host genetic determinants. Due to the sporadic cases of this study and the paucity of data
currently available in literature on this issue, future investigations in a larger population are needed to confirm ourfindings.
Keywords: Hepatocellular carcinoma, Intrahepatic Cholangiocarcinoma, ABCC2 c.3972C > T polymorphism, Wilson
disease, Risk factors

BackgroundWilson disease is a rare autosomal recessive genetic disorder linked to a mutation of the copper-transporting P* Correspondence: giovanni.brandi@unibo.it
1
Division of Oncology, Azienda Ospedaliero-Universitaria of Bologna,
Bologna, Italy2
Department of Experimental, Diagnostic and Specialty Medicine, S.
Orsola-Malpighi University Hospital, via Massarenti 9, 40138 Bologna, Italy
Full list of author information is available at the end of the article

type ATPase (ATP7B) gene, encoding the ATP7B protein, the main regulator of the hepatic metabolism ofcopper in the body [1]. As a result, large amounts ofcopper deposit in the liver and, entering into the systemic circulation, finally accumulate in the brain, kidneys and other vital organs [1]. Clinical presentation canbe very heterogeneous but the pathognomonic featuresof Wilson disease are liver disease and cirrhosis, Kayser–© The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License,
which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give
appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate ifchanges were made. The images or other third party material in this article are included in the article's Creative Commonslicence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain
permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to thedata made available in this article, unless otherwise stated in a credit line to the data.

Brandi et al. BMC Medical Genetics(2020) 21:225

Fleischer rings in Desçemet’s membrane of the cornea,
neuropsychiatric symptoms and acute episodes ofhemolysis [1]. Despite copper overload is surely involvedin the early stage of liver damage, chronic inflammationand cirrhosis, its direct role in liver carcinogensis still remains to be fully clarified.
The development of hepatocellular carcinoma (HCC)and intrahepatic cholangiocarcinoma (iCCA), the twomain primary liver cancers (PLCs), is a rare event in
Wilson disease patients, in contrast with the occurrence
observed in other chronic liver diseases [2]. Conversely,
recent studies suggest a potential role of some single nucleotide polymorphisms (SNPs) in increasing PLC susceptibility. Among these, the c.3972C > T genetic variant
at exon 28 of the ABCC2 gene has been reported to significantly associate with both HCC and iCCA increased
risk [3, 4]. ABCC2 is an ATP-binding cassette (ABC)transporter involved in phase III metabolism of carcinogens and toxins and plays a critical role in cellular defence. In the liver this transporter regulates thepharmacokinetics of many drugs and xenobiotic metabolism by the excretion of conjugates carcinogens into
bile [5].
In the present study we report a familiar cluster of five
siblings who were found to carry the ABCC2 c.3972C >T genetic variant. Three of them were affected by Wilson disease and two brothers with Wilson disease also
developed HCC and/or iCCA; the remaining two siblings are currently without any evidence of Wilson disease and/or PLC development.

Methods
Risk factors assessment

The presence of known risk factors for HCC andiCCA in the five siblings was investigated after an accurate anamnesis of their clinical history. Information
on cigarette smoking was evaluated by a questionnaire, as previously reported [6]. Subjects smoking≤100 cigarettes (the equivalent of 5 packs) in their
lifetime were classified as never smokers; subjects
smoking > 100 cigarettes in their lifetime were classified as current or former smokers, depending onwhether they had smoked cigarettes in the past 30
days. Duration of smoking (years) was calculated asfollows: present age - age of smoking onset for
current smokers; age at quitting cigarettes - age ofsmoking onset for former smokers. Subjects were also
asked about the mean number of cigarettes/day. Packyears was calculated as follows: number of cigarette
packs/day x number of years smoking.
Information on asbestos exposure was assessed according to National Registry of Mesotheliomas (ReNaM)
questionnaire, as previously reported [7].

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Genomic DNA extraction and sanger sequencing

Genomic DNA was extracted from peripheral blood
lymphocytes of the five siblings using the QIAampR
DNA Blood Maxi Kit (QIAGEN, Valencia, CA, USA),according to the manufacturer’s protocol. For detectionof c.3972C > T SNP at exon 28 in ABCC2 gene (Reference Sequence NM_000392.4), PCR amplification was
performed with the following primer sequences: Forward: 5′-CACTGCTACCCTTCTCCTGT-3′; Reverse:
3′-GACCCTTTCCCTCCATCCAA-5′.
Amplification
conditions were 95 °C for 10 min, 35 cycles at 95 °C for
30s, 60 °C for 30s, 72 °C for 45 s, and finally 72 °C for 7
min. Purified PCR products were then sequenced withBig Dye v1.1 (Life Technologies) according to manufacturer’s instructions and the reaction products analyzed
on 3730 DNA Analyzer (Life Technologies, Carlsbad,
CA, USA).

Results
In December 2013 a 56-year-old male patient presented
at our center with a significant weight loss; a contrast enhanced computed tomography (CT) scan revealed a hepatic lesion measuring 35 × 22 mm in diameter,
occupying segment IV. The patient received genetic diagnosis of Wilson disease when he was 43 years old. Bloodtests excluded HBV and HCV infection and no evidenceof non-alcoholic fatty liver disease/non-alcoholic steatohepatitis was identified by histological examination ofhepatic biopsy. The patient reported a history of > 45
pack-year of cigarette smoking and a job as railroader.
According to standardized questionnaires [6, 7], he resulted an active heavy smoker and moderately exposed
to asbestos. The patient underwent wedge resection ofsegment IV and the histological examination was compatible with iCCA, moderately differentiated (G2). In
May 2014, a CT scan demonstrated a new focal lesion in
the segment VII and another wedge resection was performed. Histology revealed HCC (G1, R0, N0). He
remained without evidence of disease for over 5 years.
The second case refers to the brother, who received
genetic diagnosis of Wilson disease when he was 36 years
old. Fifteen years later, in December 2016, an abdominal
ultrasound revealed the presence of four hypoechoic
liver lesions occupying segments II, V, VI and VII-VIII,
confirmed by a contrast-enhanced ultrasound (CEUS)
exam and a CT scan. A biopsy of the VII-VIII segment
lesion was performed and histology was consistent withwell-differentiated (G1) iCCA. In January 2017, the patient underwent surgery, with no metastatic lymph
nodes and radical margins. Post-operative histologicalexamination confirmed the diagnosis of welldifferentiated (G1) iCCA. In May 2017, the patient wasreferred to our centre for the first time and we decided
to start a follow-up program; given the time elapsed

Brandi et al. BMC Medical Genetics(2020) 21:225

between surgery and our clinical evaluation, we excluded
adjuvant chemotherapy. In January 2018, CEUS, FDGPET and abdominal MRI detected at least six focal hepatic lesions, suspected for disease recurrence. In a multidisciplinary approach, orthotopic liver transplantation(OLT) was considered only after a conversion treatment
strategy using Gemcitabine-Cisplatin schedule. We
started the treatment in February 2018 withGemcitabine-Cisplatin for a total of 4 cycles; in June
2018, the radiological reassessment via CT-PET and
MRI revealed partial response, with significant numericaland dimensional decrease of the target lesions. Twomonths later, the patient underwent OLT; 1 year after
OLT, he is without evidence of disease. Similarly to thebrother, the patient reported a history of > 50 pack-yearof cigarette smoking and worked as railroader, resulting
an active heavy smoker and moderately exposed to asbestos, according to standardized questionnaires [6, 7].
No other risk factors linked to iCCA development [8]emerged from the clinical history.
Due to the extreme rarity of PLC occurrence in two
brothers affected by Wilson disease, we hypothesizedthat further risk factors may be involved in cancer development. Basing on their history of active heavy smokers

Page 3 of 6

and moderate exposure to asbestos, as well as on the reported link between increased PLC risk and the ABCC2
c.3972C > T SNP [3], the presence of this genetic variant
involved in xenobiotic metabolism was investigated in
the brothers. As shown in Fig. 1 (Cases 1 and 2), theyboth resulted carriers of the ABCC2 c.3972C > T SNP.
Notably, also the other three siblings resulted positive
for this SNP (Cases 3, 4 and 5). Among these three siblings, only a sister received genetic diagnosis of Wilson
disease when she was 31 years old, while the others had
no evidence of Wilson disease and/or PLCs (Fig. 2). The
father died of liver cirrhosis, although it was not possible
to determine if related to Wilson disease or not. Unfortunately, neither the mother nor other relatives wereavailable for genetic testing and therefore we were unable
to assess the segregation of Wilson disease and ABCC2
c.3972C > T variant in the family.

Discussion
In the present study we report the clinical case of five
siblings who were found to carry the ABCC2 c.3972C >T SNP; among them, three were affected by Wilson disease and two brothers with Wilson disease also developed PLCs during their life.

Fig. 1 DNA Sanger sequencing from the peripheral blood lymphocytes of the five siblings (Cases 1–5), showing the presence of c.3972C > T SNP
at exon 28 of ABCC2 gene in all the samples. SNP: single nucleotide polymorphism; Ref Seq: reference sequenceBrandi et al. BMC Medical Genetics(2020) 21:225

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Fig. 2 Family pedigree. Type of diseases are shown in the legend. Black arrows show the two brothers with concomitant c.3972C > T SNP atexon 28 of ABCC2 gene, Wilson disease and primary liver malignancies. SNP: single nucleotide polymorphism; WD: Wilson disease; HCC:
hepatocellular carcinoma; iCCA: intrahepatic cholangiocarcinoma

The occurrence of PLCs in Wilson disease patients is
considered a rare event - especially in iCCA - and theunderlying molecular mechanisms have not been yet fully
clarified. Sporadic cases of HCC have been described inliterature, especially in male subjects, while the occurrenceof iCCA is even rarer [9]; moreover, to our knowledge, thedevelopment of HCC and iCCA in a patient with Wilson
disease has never been reported so far.
A recent multicenter cohort study analyzed 1186 patients affected by Wilson disease; the incidence of PLCs
was 0.28 per 1000 person years, with a prevalence of
1.2% [10]. Moreover, the incidence of HCC and iCCA
for individuals with Wilson disease have been found tobe 0.7 and 0.5%, respectively. These incidences are extremely low if compared to those ones occurring inother bile duct disorders and liver diseases, such as primary sclerosing cholangitis, cirrhosis, viral hepatitis andhepatolithiasis [8]. Currently only sixteen cases of iCCA
associated with Wilson disease have been reported in literature, including the two patients of the present study(Supplementary Table 1). Interestingly, although theoverall low number of patients precludes the possibilityof making strong statements regarding gender differences, a slight prevalence of male subjects has been observed (62.5%, 10/16 cases reported). Previous studies
about iCCA in Wilson disease are mainly case reports
and case series on a very small number of patients. Sperling et al. reported a patient undergoing liver transplantation for Wilson disease [11], while Saito et al. describeda diagnosis of iCCA following a right hepatic lobectomy
in a patient with Wilson disease [12]. Similarly, HCC isconsidered a rare finding in Wilson disease, with approximately forty cases reported in literature. In a recent
multicenter Dutch series, after a median follow up of 15
years, only 2 cases of HCC were reported in 130 patientsaffected by Wilson disease, with an annual HCC risk of
0.14% for cirrhotic subjects and 0.09% for all patients[13]. From a biological point of view, the protective roleof copper against cancer development is still debated. Indeed, while an excess of copper is known to induce

DNA damage via reactive oxygen species (ROS) generation [14], a protective role of this metal against cancer
development has been also reported [15]. Althoughchronic liver damage is likely the most important risk factor for PLCs onset, the rarity of these tumours in Wilson
disease patients suggests that further host/exogenous factors are likely involved in liver carcinogenesis.
In line with this hypothesis, the two brothers developing PLCs shared a moderate exposure to asbestos and ahistory of heavy active cigarette smoking. Conversely, norisk factors potentially linked to HCC and iCCA (including exposure to environmental carcinogens such as smoking and asbestos) emerged from the clinical history of
the other three siblings. As to asbestos, a not-negligible
increased risk for PLCs, particularly for iCCA, has been
recently reported in workers occupationally exposed tothis compound [16–18]. For cigarette smoking, some recent meta-analyses reported a moderate association with
PLCs risk [19], even if the effective role in the onset ofthese diseases still remains to be fully clarified. It is
worth to underline, however, that in the two brothers
developing PLCs, a moderate exposure to asbestos and ahistory of active cigarette smoking co-occurred with thepresence of the ABCC2 c.3972C > T genetic variant.
Notably, this variant has been detected with more frequency in iCCA patients compared to healthy population (39.2% vs 26.0%, p = 0.022) with an odds ratio = 1.83
(95% CI 1.09–3.08) [3]; similarly, this variant has beensignificantly associated with increased HCC risk in astudy comparing 53 patients and 57 healthy controls [4].
In the liver the ABCC2 transporter is expressed on themembrane of both hepatocytes and cholangiocytes andplays a critical role in xenobiotic metabolism by biliary
excretion of toxins and carcinogens [20]; in particular, it
regulates the metabolism of the 4-(methylnitrosamino)1-(3-pyridyl)-1-butatone, one of the most toxic procarcinogens found in tobacco [21]. Although the ABCC2
c.3972C > T SNP identified in the five siblings was a synonymous genetic variant not inducing changes in theamino acid sequence of the protein, it may significantlyBrandi et al. BMC Medical Genetics(2020) 21:225

affect protein function of absorption/excretion of toxins
and carcinogens due to aberrant folding, similarly towhat reported for a synonymous SNP occurring in themulti-drug resistance 1 gene [22]. It could be therefore
hypothesized that in the two brothers with Wilson disease and a history of heavy active smokers and moderate
exposure to asbestos, the presence of ABCC2 c.3972C >T genetic variant may have lead to an increased concentration of these carcinogens in the liver tissue over theyears, thereby promoting PLC development. Conversely,
the lack of PLC development in the three siblings with
ABCC2 c.3972C > T SNP alone or in association with
Wilson disease, but not exposed to these carcinogens
and other known risk factors for PLC, suggest that these
two genetic conditions may be not sufficient for PLC development, but that co-occurrence of further host/exogenous risk factors are likely needed to drive thisprocess.

Conclusions
Overall these findings reinforce the notion that liver carcinogenesis is the result of a complex interplay between
environmental and host genetic determinants. However,
because of the sporadic cases analyzed in this study andthe paucity of data currently available in the literatureon this issue, future investigations in a larger populationof subjects are needed to confirm our findings.
Supplementary Information
The online version contains supplementary material available at https://doi.
org/10.1186/s12881-020-01165-0.
Additional file 1:Supplementary Table 1: Published studies about ICC
development in WD patients [23–25].
Additional file 2:Supplementary file including ABCC2 genesequencing raw data.

Abbreviations
HCC: Hepatocellular carcinoma; iCCA: Intrahepatic cholangiocarcinoma;
PLC: Primary liver cancer; SNP: Single nucleotide polymorphisms;
CT: Computed tomography; CEUS: Contrast-enhanced ultrasound;
OLT: Orthotopic liver transplantation
Acknowledgments
The authors thank Dr. Luciana Gargano for technical support.
Authors’ contributions
GB, AR, ST: conception of the study, data analysis and article drafting; GB, FT,
AP and SDL: patients management and critical revision of the manuscript;
MD and VR: patients questionnaires and risk factors analysis; VI, SB and MP:
molecular analysis. All the authors critically revised the article and approved
the final version to be published.
Funding
The costs for molecular analysis of the present study were supported by
Fondazione Cassa di Risparmio di Bologna (grant ID: 2015/0300) to Prof. G.
Brandi.

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Availability of data and materials
All genomic data generated or analysed during this study are available assupplementary file. ABCC2 reference sequence is available at https://www.
ncbi.nlm.nih.gov/nuccore/NM_000392.4 web site.
Ethics approval and consent to participate
The present study has been approved by the local ethical committee of
S.Orsola-Malpighi University Hospital (Register 21/2014/U/Tess). Written
informed consent to participate at the study was obtained from the patientsin accordance with institutional guidelines, including the Declaration of
Helsinki (and subsequent modifications).
Consent for publication
Written informed consent for publication of the results obtained in present
study was obtained from the patients.
Competing interests
The authors declare no competing interests.
Author details
Division of Oncology, Azienda Ospedaliero-Universitaria of Bologna,
Bologna, Italy. 2Department of Experimental, Diagnostic and Specialty
Medicine, S. Orsola-Malpighi University Hospital, via Massarenti 9, 40138
Bologna, Italy. 3Center for Applied Biomedical Research, S. Orsola-Malpighi
University Hospital, Bologna, Italy. 4”Giorgio Prodi” Cancer Research Center,
University of Bologna, Bologna, Italy. 5Department of Medical and Surgical
Sciences, S. Orsola-Malpighi University Hospital, Bologna, Italy.
1

Received: 29 May 2020 Accepted: 9 November 2020

References
1. European Association for Study of Liver. EASL clinical practice guidelines:
Wilson's disease. J Hepatol. 2012;56(3):671–85.
2. Walshe JM, Waldenstrom E, Sams V, Nordlinder H, Westermark K. Abdominal
malignancies in patients with Wilson's disease. QJM. 2003;96(Suppl 9):657–
62.
3. Hoblinger A, Grunhage F, Sauerbruch T, Lammert F. Association of the c.
3972C>T variant of the multidrug resistance-associated protein 2 Gene
(MRP2/ABCC2) with susceptibility to bile duct cancer. Digestion. 2009;80:36–
9.
4. Meyer-Heithuis C, Krone B, Ehmer U, Manns MP, Strassburg CP. Analyse vonPolymorphismen des cMRP2-Gens (ABCC2) von Patienten mit
hepatozellulärem Karzinom (HCC). Z Gastroenterol. 2004;42:931.
5. Jedlitschky G, Hoffmann U, Kroemer HK. Structure and function of the MRP2(ABCC2) protein and its role in drug disposition. Expert Opin Drug Metab
Toxicol. 2006;2:351–66.
6. Al Rifai M, DeFilippis AP, McEvoy JW, et al. The relationship between
smoking intensity and subclinical cardiovascular injury: the multi-ethnic
study of atherosclerosis (MESA). Atherosclerosis. 2017;258:119–30.
7. Brandi G, Deserti M, Palloni A, et al. Intrahepatic cholangiocarcinoma
development in a patient with a novel BAP1 germline mutation and low
exposure to asbestos. Cancer Genet. 2020;S2210–7762(20):30276–3.
8. Khan SA, Tavolari S, Brandi G. Cholangiocarcinoma: epidemiology and risk
factors. Liver Int. 2019;39(Suppl 1):19–31.
9. Xu R, Hajdu CH. Wilson disease and hepatocellular carcinoma. Gastroenterol
Hepatol (N Y). 2008;4(6):438–9.
10. Pfeiffenberger J, Mogler C, Gotthardt DN, et al. Hepatobiliary malignancies
in Wilson disease. Liver Int. 2015;35(5):1615–22.
11. Sperling J, Justinger C, Schuld J, Ziemann C, Seidel R, Kollmar O.
Intrahepatic cholangiocarcinoma in a transplant liver--selective internalradiation therapy followed by right hemihepatectomy: report of a case.
World J Surg Oncol. 2014;12:198.
12. Saito T, Watanabe M, Tanaka H, Asai K, Osawa A, Matsukiyo H, Kusachi S,
et al. Cholangiocellular carcinoma associated with Wilson’s disease. Jpn J
Gastroenterol Surg. 2009;42:1574–9.
13. van Meer S, de Man RA, van den Berg AP, et al. No increased risk ofhepatocellular carcinoma in cirrhosis due to Wilson disease during longterm follow-up. J Gastroenterol Hepatol. 2015;30(3):535–9.

Brandi et al. BMC Medical Genetics(2020) 21:225

14. Angelé-Martínez C, Goodman C, Brumaghim J. Metal-mediated DNA
damage and cell death: mechanisms, detection methods, and cellularconsequences. Metallomics. 2014;6(8):1358–81.
15. Wilkinson ML, Portmann B, Williams R. Wilson's disease and hepatocellular
carcinoma: possible protective role of copper. Gut. 1983;24(8):767–71.
16. Brandi G, Di Girolamo S, Farioli A, et al. Asbestos: a hidden player behindthe cholangiocarcinoma increase? Findings from a case-control analysis.
Cancer Causes Control. 2013;24(5):911–8.
17. Farioli A, Straif K, Brandi G, et al. Occupational exposure to asbestos and riskof cholangiocarcinoma: a population-based case-control study in four
Nordic countries. Occup Environ Med. 2018;75(3):191–8.
18. Brandi G, Tavolari S. Asbestos and intrahepatic Cholangiocarcinoma. Cells.
2020;9(2):421.
19. Petrick JL, Campbell PT, Koshiol J, et al. Tobacco, alcohol use and risk ofhepatocellular carcinoma and intrahepatic cholangiocarcinoma: the liver
Cancer pooling project. Br J Cancer. 2018;118(7):1005–12.
20. Dietrich CG, Geier A, Oude Elferink RP. ABC of oral bioavailability:transporters as gatekeepers in the gut. Gut. 2003;52(12):1788–95.
21. Hecht SS. Carcinogen derived biomarkers: applications in studies of human
exposure to secondhand tobacco smoke. Tob Control. 2004;13(Suppl 1):i48–
56.
22. Kimchi-Sarfaty C, Oh JM, Kim IW, et al. A "silent" polymorphism in the MDR1
gene changes substrate specificity. Science. 2007;315(5811):525–8.
23. Mavilia MG, Sharma A, Forouhar F. Metastatic cholangiocarcinoma in apatient with undiagnosed Wilson disease. Ann Hepatol. 2019;18(6):929–31.
24. Németh D, Folhoffer A, Smuk G, Kajtár B, Tornóczky T, Szalay F.
Cholangiocarcinoma in Wilson's disease - a case report. J Gastrointestin
Liver Dis. 2017;26(3):305–8.
25. Mukai Y, Wada H, Eguchi H, et al. Intrahepatic cholangiocarcinoma in apatient with Wilson's disease: a case report. Surg Case Rep. 2016;2(1):29.

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